Ambient Temperature Stable Kits for Molecular Diagnostics

Abstract

A method for processing DNA polymerase and/or dNTPs for use in an amplification procedure, includes providing a solution mixture, the solution mixture including a DNA polymerase and/or dNTPs, a buffer solution and at least one stabilizing agent and hydration reducing the solution mixture. The solution mixture is hydration reduced at a temperature between 0 °C. and about 100 ° C.

1 . A method for processing DNA polymerase and/or dNTPs for use in an amplification procedure, the method comprising: 
 providing a solution mixture, the solution mixture including a DNA polymerase and/or dNTPs, a buffer solution and at least one stabilizing agent; and    hydration reducing the solution mixture, wherein the provided solution mixture is hydration reduced at a temperature between 0° C. and about 100° C.    
     
     
         2 . The method of  claim 1  further comprising: 
 storing the hydration reduced solution mixture at ambient room temperature for up to 24 months;    rehydrating the stored hydration reduced solution mixture; and    performing an amplification procedure using the rehydrated hydration reduced solution mixture.    
     
     
         3 . The method of  claim 1  wherein the DNA polymerase is a thermophilic DNA polymerase.  
     
     
         4 . The method of  claim 1  wherein hydration reducing the solution mixture comprises heating the solution mixture in an oven at a temperature of about 55° C.  
     
     
         5 . The method of  claim 1  wherein the solution mixture is hydration reduced between 50 percent and 100 percent.  
     
     
         6 . The method of  claim 1  wherein the at least one stabilizing agent comprises at least one sugar and at least one protein.  
     
     
         7 . The method of  claim 6  wherein the sugar comprises a non-reducing sugar and the protein is BSA.  
     
     
         8 . The method of  claim 7  wherein the non-reducing sugar comprises sucrose, the sucrose in a final concentration range from 1-20% and wherein the BSA concentration range is 0.5-3 mg/ml.  
     
     
         9 . The method of  claim 1  wherein the solution mixture further comprises any one or more ingredients selected from a group consisting of a set of two oligonucleotide primers, said oligonucleotide primers differing in sequence from each other; magnesium chloride; a water-soluble dye; a nucleic acid template and a fluorescent dye.  
     
     
         10 . A kit for the amplification of a nucleic acid, said kit comprising a hydration reduced solution comprising a thermophilic DNA polymerase and dNTPs, a buffer solution, at least one stabilizing agent, magnesium chloride, a set of two oligonucleotide primers, said oligonucleotide primers differing in sequence from each other and an oligonucleotide probe that differs in sequence from said set of two oligonucleotide primers, with or without a nucleic acid template.  
     
     
         11 . A kit for the amplification and detection of nucleic acids, said kit comprising: 
 a. a solution comprising a thermophilic DNA polymerase and dNTPs, a buffer solution, at least one stabilizing agent, magnesium chloride, a set of two oligonucleotide primers, said oligonucleotide primers differing in sequence from each other,    b. at least one additional set of oligonucleotide primers, said oligonucleotide primers differing in sequence from each other and being capable of amplifying a region of target DNA that is distinct from the region of target that may be amplified by the first set of oligonucleotide primers,    wherein the reagents of the kit are hydration reduced by means of drying at elevated temperatures, lyophilization, vacuum hydration removal, spray drying, fluidized bed drying or drum drying, and wherein the kit reagents are capable of nucleic acid amplification after having been stored at ambient temperature for up to 90 days and subsequently rehydrated.    
     
     
         12 . The kit of  claim 11  which further includes at least one oligonucleotide probe that differs in sequence from said first and second set of oligonucleotide primers.  
     
     
         13 . The kit of  claim 11  wherein the reagent components are pre-loaded into a single PCR reaction microtube, preloaded into a PCR reaction microtube that is part of a microtube strip or preloaded into a well of a multi-well plate prior to the hydration reduction of the reagents being reduced.  
     
     
         14 . The kit of  claim 13  wherein the hydration reduced solution further includes a water-soluble dye and wherein the amplification process is PCR.  
     
     
         15 . The kit of  claim 12  wherein the reagent components are pre-loaded into a single PCR reaction microtube, preloaded into a PCR reaction microtube that is part of a microtube strip or preloaded into a well of a multi-well plate prior to the hydration reduction of the reagents being reduced  
     
     
         16 . The kit of  claim 15  wherein the hydration reduced solution further includes a fluorescent dye, wherein the oligonucleotide probe or probes are labeled with a detectable label moiety and the amplification process is quantitative PCR.  
     
     
         17 . The kit of  claim 16  wherein one oligonucleotide probe is capable of quantitatively detecting the amplification of a target sequence and at least one additional oligonucleotide probe is capable of quantitatively detecting the amplification of a different target sequence.  
     
     
         18 . A kit for the amplification and detection of nucleic acids, the kit comprising: 
 a. a first set of two oligonucleotide primers, one or both of which is fluorescent labeled and/or an additional third oligonucleotide that is a labeled probe, said oligonucleotide primers and probe differing in sequence from each other, and able to detect in a quantitative PCR reaction the presence of a unique nucleic acid sequence,    b. a second set of oligonucleotide primers, one or both of which is fluorescent labeled to serve as a probe or in additional to a third oligonucleotide serving as a probe, said second set of oligonucleotide primers and probe differing in sequence from each other, and able to detect in a quantitative PCR reaction the presence of a distinct nucleic acid sequence that is different from the nucleic acid sequence being detected by the first set of primers and probe,    c. a DNA polymerase enzyme;    d. dNTPs (dATP, dCTP, dGTP and dTTP);    e. a buffer solution containing one or more stabilizing agents; and    f. magnesium chloride,    g. with or without a DNA template to serve as an internal control,    wherein at least some of the reagents of the kit, including the DNA polymerase enzyme and the dNTPs, are hydration reduced by means of drying at elevated temperatures, lyophilization, vacuum hydration removal, spray drying, fluidized bed drying or drum drying and wherein the kit reagents together in a single mixture are capable of nucleic acid amplification activity after having been stored at ambient temperatures for up to 90 days and subsequently rehydrated.    
     
     
         19 . The kit of  claim 18  further comprising at least one additional set of oligonucleotide primers and probes, wherein the at least one additional set of oligonucleotide primers and probes is capable of performing a multiplex PCR reaction.  
     
     
         20 . The kit of  claim 18  wherein one or more of the oligonucleotides is single labeled with a fluorescent moiety to serve as a detection probe or dual-labeled as a fluorescence resonance energy transfer (FRET) probe.  
     
     
         21 . The kit of  claim 18  wherein magnesium chloride is not included in the solution prior to hydration reduction and wherein magnesium chloride is later added to the solution to facilitate nucleic acid amplification.